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Abstract:

A delivery device for an active agent comprises nanoparticles based on a
biopolymer such as starch. The delivery device may also be in the form of
an aptamer-biopolymer-active agent conjugate wherein the aptamer targets
the device for the treatment of specific disorders. The nanoparticles may
be made by applying a high shear force in the presence of a crosslinker.
The particles may be predominantly in the range of 50-150 nm and form a
colloidal dispersion of crosslinked hydrogel particles in water. The
biopolymer may be functionalized. The aptamer may be conjugated directly
to the cross-linked biopolymers. The active agent may be a drug useful
for the treatment of cancer. The delivery device survives for a period of
time in the body sufficient to allow for the sustained release of a drug
and for the transportation and uptake of the conjugate into targeted
cells. However, the biopolymer is biocompatible and resorbable.

Claims:

1. A medicament comprising, a) nanoparticles comprising a mass of
crosslinked polymers, at least 50% of the polymers being high molecular
weight starch; and, b) active agent molecules conjugated with the
nanoparticles.

2. The medicament of claim 1 wherein the nanoparticles have a number
average size in the range of 50-150 nm when measured by any of SEM, NTA
or DLS.

3. The medicament of claim 2 wherein most of the nanoparticles have a
size in the range of 50-150 nm when measured by any of SEM, NTA or DLS.

4. The medicament of claim 1 wherein the active agent comprises a drug.

5. The medicament of claim 4 wherein the drug is a chemotherapeutic drug.

6. The medicament of claim 1 wherein the zeta potential of the
nanoparticles is negative.

7. The medicament of claim 1 wherein the nanoparticles further comprises
ligands conjugated to the cross-linked starch polymers.

8. The medicament of claim 7 wherein the ligands are aptamers.

9. A method of making a medicament comprising the steps of, a) forming a
plurality of nanoparticles, the nanoparticles comprising a mass of
crosslinked polymers, at least 50% of the polymers being amylose,
amylopectin, or a mixture of amylose and amylopectin; and, b) combining
an active with the nanoparticles.

10. The method of claim 9 further comprising a step of functionalizing
the nanoparticle such that the nanoparticle has a zeta potential of
negative 10 mV or a more negative zeta potential.

19. The compound of claim 18 wherein the aptamer is attached to the
starch.

20. The compound of claim 19 wherein the aptamer has an amine used in
attaching the aptamer to the starch.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] For the United States of America, this application claims the
benefit under 35 USC 119 of U.S. Provisional Application No. 61/419,106
filed on Dec. 2, 2010 which is incorporated by reference.

FIELD

[0003] This specification relates to a delivery device for drugs or other
agents, to methods of making and using the delivery device, and to the
treatment of cancer.

BACKGROUND

[0004] The following discussion is not an admission that anything
described below is common general knowledge.

[0005] U.S. Pat. No. 6,340,527 to Van Soest et al. describes
microparticles having a particle size of 50 nm to 1 mm consisting of a
chemically cross-linked starch shell containing an active ingredient. The
particles are obtained by first preparing an oil in water emulsion of the
active ingredient in a hydrophobic phase and starch, or a dispersion of a
solid active ingredient and starch in water. The active ingredient may be
a medicament which is released in the digestive tract when the starch
degrades.

[0006] US Patent Application Publication US 2008/0241257 to Popescu et al.
describes a nanoparticle of a biodegradable polymer containing a
hydrophilic cationic drug such as streptomycin. The biodegradable polymer
may be chitosan. A pharmaceutical preparation containing the
nanoparticles is administered to a patient orally and the nanoparticles
release the drug in vivo. The drug can be complexed with a naturally
occurring polymer, such as dextran sulfate. The drug, optionally
complexed, is mixed with the biodegradable polymer followed by an
inorganic polyanion to form the nanoparticle. In one example, the
nanoparticles were about 560 nm in average size, had a zeta potential of
about +54 mV and were used to treat tuberculosis in mice.

[0007] U.S. Pat. No. 7,550,441 to Farokhzad et al. describes a conjugate
that includes a nucleic acid ligand bound to a controlled release polymer
system contained within a pharmaceutical compound. Some examples of the
polymer system are based on poly(lactic) acid (PLA) and have mean
particle sizes ranging from 137 to 2805 nm. The ligands have an affinity
for a target and are prepared through the Systemic Evolution of Ligands
by Exponential Enrichment (SELEX) process.

[0008] US Patent Publication 2009/0312402 to Contag et al. describes
nanoparticles with encapsulated nucleic acid. The polymer may be PLA, PLG
or PLGA and PEG. The particles may have ligands or antibodies attached to
them for targeting the nanoparticles to a site of interest. The
nanoparticles may have a polymer coating to provide controlled release.
The particles are in the size range of about 50 nm to about 500 nm, with
most of them in the sub-200 nm range.

[0009] US Patent Publication 2011/0244048 to Amiji et al. describes a
method of making a nanoparticle comprising combining an aqueous solution
of a solubilized therapeutic agent with a water-soluble polymer
comprising polyethylene glycol (PEG) and a fatty acid. These components
self assemble into a nanoparticle. Various dextran based particles have
means sizes ranging from 14 nm to 430 nm. The therapeutic agent may be
doxorubicin.

[0010] U.S. Pat. No. 8,048,453 to Sung et al. describes nanoparticles of
chitosan, poly-glutamic acid, and an active agent. The particles have a
mean particle size between about 50 nm and 400 nm. The active agent may
be insulin for the treatment of diabetes or an active for treating
Alzheimer's disease. The nanoparticles may be freeze-dried and loaded
into a capsule for oral administration.

INTRODUCTION TO THE INVENTION

[0011] The following introduction is intended to introduce the reader to
the invention and the detailed description to follow and not to limit or
define the claims.

[0012] This specification describes a nanoparticle based delivery device.
The device may be used for the treatment of various indications or for
other purposes. However, this specification will primarily describe the
use of the device to deliver chemotherapeutic drugs to treat cancer.

[0013] The development of new and improved chemotherapeutic delivery
devices is clearly important. Cancer is the second most common cause of
death in the US, accounting for 1 of every 4 deaths. With solid tumor
cancers, sufficiently localized tumors can be removed by a surgeon. In
most cases, however, not all of the tumor is removed and follow-up
therapy with radiation or chemotherapy is required. In the United States,
about 50% of cancer patients will receive chemotherapy. With metastatic
cancers that are widely disseminated upon diagnosis, such as leukemias,
chemotherapy is required.

[0014] The delivery device described in this specification includes a
nanoparticle that was originally developed as an industrial latex. In
brief, the particle is made predominantly from a biopolymer, such as a
starch comprising amylose or amylopectin or both, that has its crystal
structure broken, for example by shear forces and intensive mixing in the
presence of a hydroxilic solvent. After the crystal structures have been
broken, a crosslinking agent is added. The resulting nanoparticles,
comprising for example cross-linked high molecular weight starch
polymers, can be handled as dry agglomerated particles and later
dispersed in an aqueous medium to produce a stable latex dispersion of
crosslinked hydrogel nanoparticles.

[0015] These particles have been used, for example, in paper-making
slurries, as a binder in a pigmented paper coating composition, and as an
adhesive. However, the inventors believe that these particles have
attributes that make them useful as a drug delivery device. In an aqueous
medium, the nanoparticles form a stable dispersion of swollen crosslinked
biopolymer hydro-colloid particles. The particles swell by taking water
into the core of the particle. This mechanism may be used to load a drug
into the core of the particle while allowing the drug to be released
later in the body of a person, or another mammal. The nanoparticles can
be administered as a liquid suspension or dried to produce a powder.

[0016] One useful attribute of the nanoparticles is that they can be
broken down by chemical and enzymatic elements, but they persist in the
body long enough to give a sustained drug release. While native starch
particles would survive for less than 30 minutes in the body, the
crosslinked starch nanoparticles have a considerably longer half life. In
a related attribute, the nanoparticles can provide two mechanisms for
releasing an encapsulated drug. According to a first mechanism, the drug
is released from a generally intact particle. According to a second
mechanism, degradation of the particle releases more of the drug. This
provides a sustained release of the drug, which is useful for therapeutic
agents that require several hours or more of residence time within the
body for the drug to act.

[0017] Another attribute of the nanoparticles is that the biopolymers are
compatible with the body and ultimately resorbable. The biopolymers and
their metabolites are non toxic and recognized by the body as foodstuff.
In contrast, some synthetic polymers can cause side effects when used as
a drug delivery device. For example, polyanhydride copolymers used for
drug delivery have have been associated with tissue inflammation and an
enhanced rate of infections, possibly because they degrade via hydrolysis
and yield acidic functionalities. Starch, however, is ordinarily a food
source and can be taken up by the body as it degrades essentially without
complications.

[0018] Another useful attribute of the nanoparticles is their size, and
their narrow particle size distribution which indicates a narrow range of
particles sizes within a sample. In particular, the nanoparticles have
sizes that are predominantly in the range of 50-150 nm. Particles outside
of this size range are quickly removed from the body through capillary
wall passage or by the reticuloendothelial system (RES).

[0019] Yet another useful attribute of the nanoparticles is that the
biopolymers may be functionalized. For example, amylose and amylopectin
molecules may be oxidized and provided with carboxyl functionalities. In
this example, the functionalized particles have a more negative zeta
potential which aids in the adsorption of some drugs and the attachment
of targeting moieties such as a ligand. For example, a nucleotide such as
an aptamer may be attached, for example via a carbodimide linkage,
directly to the surface of a crosslinked structure forming the core of
the particle. Other forms of functionalization may influence the
attachment of a targeting molecule or the release profile of a drug.

[0020] A drug delivery device may take advantage of any one or more of
these or other attributes. An example of a drug delivery device described
in this specification comprises nanoparticles made predominantly of high
molecular weight starch or cross-linked biopolymers and conjugated to an
active agent such as a chemotherapeutic drug. The nanoparticles may be
made by mixing under high shear forces and then adding a crosslinker. The
nanoparticles are predominantly in the range of 50-150 nm in size and
form a hydro-colloidal dispersion in water. Optionally, the
nanoparticles, and in particular the crosslinked polymers, may be further
conjugated to a targeting molecule such as an aptamer. The aptamer
targets the nanoparticles for delivery of the active agent to cancer
cells.

[0021] A drug delivery device may have: 1) a nanoparticle comprising
crosslinked biocompatible or resorbable polymers, the polymers modified
after the particle was formed by chemical or enzymatic modification, 2)
an encapsulated active agent within the colloidal hydrogel, and,
optionally, 3) an aptamer attached to the cross-linked polymers. The
nanoparticles may be colloidal hydrogel particles.

[0022] A medicament described in this specification comprises a plurality
of nanoparticles, the nanoparticles made up mostly of high molecular
weight starch, with an active agent conjugated to at least some of the
nanoparticles. The medicament may be useful in the treatment of cancer. A
method of making a medicament comprises steps of forming a plurality of
high molecular weight starch based nanoparticles, the nanoparticles
having a size predominantly in the range of 50 to 150 nm, and combining a
drug or other agent with a least some of nanoparticles.

[0023] A compound described in this specification comprises a high
molecular weight starch based nanoparticle core having a size in the
range of 50 to 150 nm, a therapeutic agent and an aptamer. The compound
may be used for the treatment of cancer.

[0029] FIG. 6 is a schematic representation of minor swelling in an SB
latex particle and significant swelling of the cross-linked starch
nanoparticles of FIG. 2 in an aqueous dispersion, illustrating the
hydrocolloid structure of the starch based nanoparticles.

[0030] FIG. 7 is a schematic model of the cross-linked starch
nanoparticles of FIG. 2.

[0031] FIG. 8 is a chart showing the fluorescence spectrum of free
Doxorubicin and Doxorubicin entrapped in the cross-linked starch
nanoparticles of FIG. 2.

[0032] FIG. 9 is a chart showing a release profile of Doxorubicin from the
cross-linked starch nanoparticles of FIG. 2.

[0033] FIG. 10 is a chart showing the fluorescence spectrum of Calcein and
a release profile of Calcein from the cross-linked starch nanoparticles
of FIG. 2.

[0034] FIG. 11 is a schematic model of a cross-linked starch nanoparticle
of FIG. 2 conjugated with a drug and an aptamer.

DETAILED DESCRIPTION

Target Particle Size

[0035] Referring to FIG. 1, particle size plays a role in determining the
fate of a drug or a drug delivery mechanism after administration.
Microparticles, being particles of one micron or greater in size, are
removed from the body within minutes by the pulmonary capillary system.

[0036] Nanoparticles (particles less than one micron in size), however,
can be rapidly removed from the blood stream by phagocyctic cells of the
reticular endothelial system (RES). (see for example Park, K.,
"Controlled Drug Delivery: Challenges and Strategies", American Chemical
Society, Washington, D.C., 1997 and Davis, S. S., "Microspheres and Drug
Therapy Pharmaceutical, Immunological and Medical Aspects", Elsevier, New
York, N.Y., USA, Chapter 2, 1984). In one study (Davis, 1984), upon
administering colloid particles of poly(styrene) latex, the particles
appeared in the Kupffer cells of the liver and subsequently in the
spleen. Leakage of latex particles into the systemic system occurred for
particles less than 100 nm, but particles of 200 to 500 nm in diameter
were removed from the blood within 5 minutes.

[0037] Particles under 70 nm in size have been shown to be taken up
through capillary wall passage and are quickly excreted by subjects.
Accordingly, extremely small particles also do not have long systemic
circulation. This includes most drugs and aptamers.

[0038] It is possible that particles in the range of about 50 to 100 nm in
size are taken up through capillary wall passage and by the RES. However,
both mechanisms may be only marginally effective against particles in
this size range and up to about 150 nm. Without intending to be bound by
any particular theory of operation, particles having a size in the range
of about 50 to 150 nm enjoy longer systemic circulation as a result of
being within this size range, independently of other properties of the
particle such as surface density or hydrophilicity which may also affect
uptake by the RES.

Biopolymer Nanoparticles

[0039] Biopolymer nanoparticles are made according to a process described
in U.S. Pat. No. 6,677,386 (which corresponds to International
Publication WO 00/69916). In the process, a biopolymer, such a starch
comprising amylose or amylopectin or both, is combined with a
plasticizer. This combination is mixed under high shear forces,
preferably in a twin screw fully intermeshing co-rotating extruder, to
plasticize the biopolymer and create a thermoplastic melt phase in which
the crystalline structure of the biopolymer is removed. A crosslinking
agent is then added while mixing continues to form cross-linked
nanoparticles. The nanoparticles exit the extruder as a strand, which is
ground to a fine dry powder. The starch based nanoparticles are present
in the powder in agglomerated form, and can be dispersed in an aqueous
medium.

[0040] The biopolymers may be starch or other polysaccharides such as
cellulose and gums, as well as proteins (e.g. gelatin, whey protein). The
biopolymers may be previously modified, e.g. with cationic groups,
carboxy-methyl groups, by acylation, phosphorylation, hydroxyalkylation,
oxidation and the like. Starch and mixtures of at least 50% starch with
other polymers are preferred. The starch, whether used alone or in a
mixture, is preferably a high molecular weight starch, for example a
molecular weight of at least 10,000, and not dextran or dextrin. For
example, the starch may be made up of amylose or amylopectin or both.
Waxy starches, such as waxy corn starch, are particularly preferred.

[0041] The following five paragraphs are repeated or summarized from U.S.
Pat. No. 6,677,386 to further describe the process of making the
nanoparticles.

[0042] The biopolymer preferably has a dry substance content of at least
50% by weight at the time when processing starts. Processing is
preferably done at a temperature of at least 40 degrees C., but below the
degradation temperature of the polymer, for example 200 degrees C. The
shear can be effected by applying at least 100 J of specific mechanical
energy (SME) per g of biopolymer. Depending on the processing apparatus
used the minimum energy may be higher; also when non-pregelatinised
material is used, the minimum SME may be higher, e.g. at least 250 J/g,
especially at least 500 J/g.

[0043] The plasticiser may water or a polyol (ethyleneglycol,
propyleneglycol, polyglycols, glycerol, sugar alcohols, urea, citric acid
esters, etc.). The total amount of plasticisers (i.e. water and others
such as glycerol) is preferably between 15 and 50%. A lubricant, such as
lecithin, other phospholipids or monoglycerides, may also be present,
e.g. at a level of 0.5-2.5% by weight. An acid, preferably a solid or
semi-solid organic acid, such as maleic acid, citric acid, oxalic,
lactic, gluconic acid, or a carbohydrate-degrading enzyme, such as
amylase, may be present at a level of 0.01-5% by weight of biopolymer.
The acid or enzyme assists in slight depolymerisation which is assumed to
be advantageous in the process of producing nanoparticles of a specific
size.

[0044] The crosslinking is preferably reversible, i.e. the crosslinks are
partly or wholly cleaved after the mechanical treatment step. Suitable
reversible crosslinkers include those which form chemical bonds at low
water concentrations, which dissociate or hydrolyse in the presence of
higher water concentrations. This mode of crosslinking results in a
temporary high viscosity during processing followed by a lower viscosity
after processing. Examples of reversible crosslinkers are dialdehydes and
polyaldehydes, which reversibly form hemiacetals, acid anhydrides and
mixed anhydrides (e.g. succinic and acetic anhydride) and the like.
Suitable dialdehydes and polyaldehydes are glutaraldehyde, glyoxal,
periodate-oxidised carbohydrates, and the like. Glyoxal is a particularly
suitable crosslinker.

[0045] Such crosslinkers may be used alone or as a mixture of reversible
crosslinkers, or as a mixture of reversible and non-reversible
crosslinkers. Thus, conventional crosslinkers such as epichlorohydrin and
other epoxides, triphosphates, divinyl sulphone, can be used as
non-reversible crosslinkers for polysaccharide biopolymers, while
dialdehydes, thiol reagents and the like may be used for proteinaceous
biopolymers. The crosslinking reaction may be acid- or base-catalysed.
The level of crosslinking agent can conveniently be between 0.1 and 10
weight % with respect to the biopolymer. The crosslinking agent may
already be present at the start of the mechanical treatment, but in case
of a non-pre-gelatinised biopolymer such as granular starch, it is
preferred that the crosslinking agent is added later on, i.e. during the
mechanical treatment.

[0046] The mechanically treated, crosslinked biopolymer is then formed
into a latex by dispersion in a suitable solvent, usually water and/or
another hydroxylic solvent such as an alcohol), to a concentration of
between 4 and 50 weight % especially between 10 and 40 wt. %. Prior to
the dispersion a cryogenic grinding step may be performed, but stirring
with mild heating may work equally well. This treatment results in a gel
which either spontaneously or after induction by water adsorption, is
broken into a latex. This viscosity behaviour can be utilised for
applications of the particles, such as improved mixing, etc. If desired,
the dispersed biopolymer may be further crosslinked, using the same or
other crosslinking agents as describe above. The extrudate is
characterised by swelling in an aqueous solvent, e.g. water or a mixture
of at least 50% water with a water-miscible solvent such as an alcohol,
and by exhibiting a viscosity drop afterwards to produce a dispersion of
nanoparticles.

[0047] International Patent Application Publication No. WO 2008/022127 A2
and its equivalent US Patent Application Publication Number 2011/0042841
A1 describe a process for producing biopolymer nanoparticles in large
quantities. US Patent Application Publication Numbers 2010/0143738 A1
describes a process for producing biopolymer nanoparticles conjugative
with additives during the extrusion process. These publications are
incorporated by reference.

[0048] The process can be operated to produce particles that have a number
average particle size in the range of 50 to 150 nm and which, considering
a distribution of their particle sizes, are also predominantly in the
range of 50 to 150 nm in size. Such particles include, for example,
EcoSphere® 2202 particles commercially available from Ecosynthetix
Inc. of Burlington, Ontario, Canada and EcoSynthetix Ltd. of Lansing,
Mich., USA. These products are made primarily from starch including
amylose and amylopectin. The product is normally sold for to replace
petroleum based latex binders in industrial applications, such as coated
paper and paperboard. The product is provided in the form of a dry powder
of agglomerated nanoparticles with a volume mean diameter of about 300
microns. When mixed in water and stirred, the agglomerates break apart
and form a stable dispersion of the nanoparticles.

[0049] Comparing FIG. 2A to FIG. 2B, the EcoSphere® 2202 nanoparticles
10 are about 100 to 300 times smaller than native starch granules 20.
Whereas a starch granule 20 may be 15 microns in size, the nanoparticles
10 are clearly well under 200 nm in size. Accordingly, the effective
surface area of the nanoparticles 10 is much greater, for example 200
square meters per gram or more.

[0050] FIGS. 3 and 4 illustrate particle size measurements of an aqueous
dispersion of EcoSphere® nanoparticles by Dynamic Laser Light
Scattering (DLS) and by Nanoparticle Tracking Analysis (NTA),
respectively. These two techniques are complementary, given that the NTA
technique is a direct measurement of the diffusion coefficient for
individual particles tracked via video tracking software (and relates
that to particle diameter via the Stokes-Einstein equation), and can
measure particles in the range of 50-1000 nm, while DLS can measure to
smaller particle sizes below 50 nm. Other techniques, including
oscillating probe Atomic Force Microscopy (AFM), Scanning Electron
Microscopy (SEM), Environmental SEM (ESEM), Transmission Electron
Microscopy (TEM) and Scanning/Transmission Electron Microscopy (STEM),
all provided similar particle size images consistent with the data in
FIGS. 3 and 4.

[0051] Referring to FIG. 3, most of the particles have a size in the range
of about 50 to 100 nm. FIG. 3 also indicates a number of particles
apparently having a size of about 10 microns. However, based on other
measurements, such as scanning electron microscope photos of freeze dried
samples as shown in FIG. 2 and the NTA measurements of FIG. 4, the
inventors believe that such larger particles may be over-represented in
the sample and that the results may include agglomerations of particles
or an anomaly in the DLS measurement. As indicated in the NTA
measurements, most of the particles (D50) are under 120 nm in size and
there are virtually no particles larger than 400 nm. Any particles larger
than 1000 nm would be removed quickly from the body causing no harm but
wasting some of an intended dosage of the drug. Accordingly, if a sample
includes material amounts of particles over 1000 nm in size, these may be
removed by filtration before a drug is loaded into the nanoparticles.

[0052] Referring to FIG. 5, the nanoparticles are generally smaller than
particles in synthetic latex emulsions The nanoparticles have a narrow
size distribution, with a polydispersity index of about 30%, and
properties characteristic of polymer colloids. The nanoparticles, since
they are predominantly in the size range of about 50 to 150 nm (for
example 50% or more of the nanoparticles by number or mass may be in this
range), the nanoparticles are cleared more slowly in the systemic system
(liver, spleen) than is the case of larger particles. The particles are
also hydrophilic, which further inhibits removal in the RES. The
degradation products of the starch nanoparticles (D-glucose and
maltodextrans) are non-toxic. The additional natural materials and
chemicals that are used to make the starch nanospheres are also
relatively non-toxic.

[0053] The discrete nanoparticles are also not water soluble, but instead
form a stable dispersion of swollen hydrogel colloidal crosslinked
particles in water.

[0054] FIGS. 6A and 6B illustrate that the biobased latex consists of
water-swollen crosslinked starch nanoparticles. They de-swell with
increasing solids so that their dispersions can be made at higher solids.
In contrast, the particles in synthetic latex emulsions do not swell nor
contain a substantial portion of water inside the colloid particles. The
swelling characteristics of typical SB latex and biolatex colloids have
been compared and reported in a number of articles (see Do Ik Lee, Steven
Bloembergen, and John van Leeuwen, "Development of New Biobased Emulsion
Binders", PaperCon2010, "Talent, Technology and Transformation", Atlanta,
Ga., May 2-5, 2010; and, Steven Bloembergen, Edward Van Egdom, Robert
Wildi, Ian. J. McLennan, Do Ik Lee, Charles P. Klass, and John van
Leeuwen, "Biolatex Binders for Paper and Paperboard Applications",
Journal of Pulp and Paper Science, 36, No 3-4, p. 151-161, 2011).

[0055] FIG. 7 illustrates a schematic model for the nanoparticles 10. The
nanoparticles 10 can be thought of as one crosslinked macromolecular
unit, with --R-- representing an intermolecular crosslink between
individual polymer 12. Other types of crosslinked structures may exist,
such as intramolecular crosslinks. The nanoparticle 10 can be thought of
as having a core 14 that takes in and releases water as it swells and
de-swells and a shell 16 which provides a steric stabilization mechanism
for the colloid particles and through which water is released, bound and
adsorbed. The structure of the nanoparticles is further described in
Steven Bloembergen, Ian. J. McLennan, John van Leeuwen and Do Ik Lee,
"Specialty Biobased Monomers and Emulsion Polymers Derived from Starch",
2010 PTS Advanced Coating Fundamentals Symposium, Munich, Germany, Oct.
11-13, 2010.

[0056] Aqueous dispersions of starch nanoparticles have been prepared that
are stable for up to 12 months or longer, compared to minutes or hours
for cooked solutions of regular corn starch or other starches. Because
typical native starches contain very high molecular weight amylopectin
polymer (millions of daltons) and high molecular weight amylose polymer
(hundreds of thousands of daltons), their solutions up to 5 or 10% solids
have very high gel-like viscosities. Commercial dispersions of corn
starch granules typically reach up to about 30% solids or higher, because
these products have been chemically, thermally or enzymatically treated
to reduce their molecular weight in order to attain higher solids
contents. This is the typical molecular weight/solids trade off that one
faces to maintain a reasonably low viscosity for polymer solutions. Much
higher solids pure dispersions (up to about 40% solids), and ultra-high
solids formulations (up to 72% solids) have been developed for
EcoSphere® starch nanospheres. This is beneficial for drug delivery
applications, where a high solids concentration in a low viscosity
dispersion facilitates high drug loadings.

[0057] The nanoparticles may be conjugated with an active agent, for
example a drug, or other agent and used as a delivery device.
Fluorescence studies indicated that the nanoparticles are taken into the
cell nucleus. Without intending to be limited by theory, the transport
mechanism is believed to be endocytosis.

[0058] As discussed above, the core of the nanoparticles takes in water as
it swells. Similarly, small molecules, other drugs, or other agents can
be taken up, adsorbed, absorbed or otherwise loaded into the core of the
nanoparticles. An example presented further below will describe the
encapsulation of doxorubicin in the nanoparticles by a phase separation
method (Example 1) and by ethanol precipitation (Example 4). By itself,
doxorubicin has been linked to acute cardiotoxicity which limits its use.
In other experiments, Carmustine and BCNU (bis(chloroethylnitrosourea))
have been loaded into the nanoparticles.

[0059] It can be expected that other methods of drug encapsulation may
also be used, and that other drugs and agents can be encapsulated. For
example, other chemotherapeutic agents such as Cyclophosphoramide and
Camptothecins might be loaded into the nanoparticle and, like
Doxorubicin, make the nanoparticles useful in the treatment of cancer.
The nanoparticles may also encapsulate non-chemotherapeutic agents, such
as antisense oligonucleotides, peptides, and cytokines for other
therapeutic applications.

[0060] After the drug is loaded, the particles can be recovered by
lyophilization. This results in a powder of the nanoparticles conjugated
with the encapsulated drug. The powder can be mixed with water, or
another hydroxylic solution, to disperse the nanoparticles. The drug can
be administered to treat a patient in this liquid form, for example
orally, by intra-venous infusion or injection. Alternatively, the powder
can be mixed with a pharmaceutical carrier and made into a solid or
gelled product, such as a tablet or capsule. The solid form may be
administered in any known manner used for pharmaceutical products, such
as orally.

[0061] The biopolymers of the nanoparticle may be modified or
functionalized through chemical or enzymatic modifications before or
after forming the nanoparticle. In principle, any chemical or enzymatic
modification known for polysaccharides can be employed. For example, a
summary of various chemical and enzymatic oxidation processes is provided
in column 1, line 66 to column 3, line 50 in R. A. Jewel et al., U.S.
Pat. No. 6,379,494, "Method of Making Carboxylated Cellulose Fibers and
Products of The Method", Apr. 30, 2002. Although these methods are
discussed in relation to cellulose, many if not all of them are adaptable
to starch polymers.

[0062] In Example 4, the starch polymers were functionalized after the
nanoparticles are formed. In particular, the polymers were oxidized to
add carboxyl functional groups. While this is described in Example 4 as
relating primarily to the attachment of an aptamer, the functionalization
was also shown to facilitate the encapsulation of doxorubicin.

[0063] The chemical or enzymatic modification may also involve other types
of functionalities introduced onto the biopolymers to provide binding
sites for the aptamer, the active agent or both. Surface modification of
the nanospheres may also alter their systemic as well as local clearance
rates to provide a better control of the delivered therapeutic dose and
the targeted delivery, if any. For example, the oxidation resulted in a
change in the zeta potential of the nanoparticle. The zeta potential of a
non-functionalized nanoparticle is in the range of 0 to negative 6 mv.
The oxidized particle had a zeta potential of about negative 25 mV. The
oxidation reaction could also be controlled to provide modified
nanoparticles having intermediate zeta potential values. Tuning the
charge on the nanoparticles will allow selective adsorption of different
drugs and agents and in addition provide a way of controlling the release
profile. Many small molecules being developed for cancer treatment are
hydrophobic and lipophilic, hence difficult to dissolve. Surface
modifications of the nanoparticle can enhance the ability of these drugs
to be loaded in the core of the nanoparticle.

[0064] While the water soluble TEMPO catalyst used in Example 4 provided
starch functionalities throughout the crosslinked nanoparticle, an
immobilized TEMPO catalyst causes only polymers at the surface of the
nanoparticle to be functionalized. This could be used, for example, to
attach an aptamer to the nanoparticle with less modification of the zeta
potential of the core of the nanoparticle.

[0065] While any form of oxidation may be used, the TEMPO oxidation is
preferred. The TEMPO catalyst is used to specifically modify the C6
hydroxyl of the glucopyranoside position to a carboxyl functionality.
This process prevents the molecular weight reduction of the
polysaccharide polymer that is common to many other oxidative processes.

[0066] Many functionalization techniques are known to add aldehyde groups
to polysaccharide polymers. Without intending to exclude the possibility
that one of these functionalization techniques might be useful, they are
not currently preferred. The aldehyde groups are reactive and tend to
cause the nanoparticles to stick together. This interferes with creating
a colloidal dispersion, and so may also interfere with distribution of
the nanoparticles in the body.

[0067] The zeta potential of unmodified nanoparticles is low, hence their
colloidal stability is attributed mainly to steric stabilization. Without
being bound by theory, the shell containing short polysaccharide chains
which project into the aqueous environment, functions as a colloidal
stabilizer for the particle in water and as a partial hydrophilic shell
of bound water. This in turn retards the efflux or diffusion of
hydrophobic drugs from the particle.

[0068] In the examples, the active agent Doxorubicin was loaded into the
nanoparticles so that the release profile could be followed using a
fluorescence technique. This work has demonstrated a biphasic release
profile with suitable release kinetics spanning multiple hours of
sustained release of the active agent. The fluorescence of the drug
loaded nanoparticle declines but some fluorescence remains even after 12
hours. This indicates that not all of drug releases from an intact
particle. The remainder of drug, however, will be released in the body as
the particle degrades, for example due to alpha-amylase enzymes in body.
The complete release time may be near 24 hours.

[0069] In animal studies described in Example 3, Doxorubicin loaded
nanospheres were used to treat glioblastoma multiforme, a primary brain
tumor in athymic mice. These studies demonstrated a 30% increase in
survival for the mice treated with doxorubicin-loaded nanospheres
relative to the appropriate controls. Without intending to limit the
invention to any particular theory, this success is attributed to one or
more of several factors including the size of the nanoparticles, the
surface properties of the nanoparticles, and the release kinetics of the
nanoparticles, for example the sustained release of the drug from the
nanoparticles. The encapsulated doxorubicin is believed to enter the cell
via endocytosis due to the relatively small size of the nanoparticle,
while the free drug is metabolized and excreted.

[0070] FIG. 11 shows a bioconjugate device 30 having an aptamer 18, a
polymer 12 and an active agent, not separately shown but provided within
the core 14. The bioconjugate device 30 may be used for the delivery of
therapeutically effective amounts of the active agent to targeted cells
for the treatment of specific disorders. The bioconjugate device 30 may
be made by making a chemical or enzymatic modification to a biocompatible
or resorbable colloidal polymer hydrogel, encapsulating an active agent
within the colloidal polymer hydrogel, and modifying the surface of the
hydrogel by attaching an aptamer onto it. The aptamer 18 typically has a
size of less than about 10 nm and increases the diameter of the
bioconjugate device 30 by only about 20 nm or less. Optionally, other
targeting ligands or other molecules might also be used.

[0071] Aptamers are capable of binding to a target molecule that are
located in a specific site which may include cancer cells. For example,
AS1411 has been shown to bind to nucleolin (Soundararajan et al., "Plasma
Membrane Nucleolin Is a Receptor for the Anticancer Aptamer AS1411 in
MV4-11 Leukemia Cells", Molecular Pharmacology, Vol. 76, No. 5, 2009).
Binding to nucleolin receptors is useful in the treatment of a wide array
of cancers such as renal cell carcinoma, breast cancer, prostate cancer
and others. AS1411 may also be tagged with, for example, a Cy3
fluorescent tag for imaging purposes.

[0072] Another potentially useful aptamer is sgc4. This aptamer was
developed by way of the SELEX process from T-cell leukemia cell lines and
is able to recognize leukemia cells (Shannguan et al., "Aptamers Evolved
from Cultured Cancer Cells Reveal Molecular Differences of Cancer Cells
in Patient Samples", Clinical Chemistry 53, No. 6, 2007). However, sgc4
has a short biological life if it is not conjugated. Its sequence is
described in US Patent Publication 2009/0117549. Shorter variants of the
sequence may also be effective. Sgc8c aptamers have also been reported to
be useful for targeting leukemia cells (Ozalp et al., Pharmaceuticals
2011, 4, 1137-1157)

[0073] Aptamers having an amine modification on the 3' end of the DNA can
be linked, for example by one or more covalent bonds, to the carboxyl
groups of the functionalized nanoparticle. The linkage may be made, for
example, using EDC chemistry or by another linkage between the carboxyl
and the amine. An example of such a linking using an amine modified test
strand of DNA is described in Example 4. Similarly, aptamers such as
AS1411 and sgc4 which can also be provided with an amine modification
should also link to the nanoparticle. When also loaded with an active
agent such as Doxorubicin, the resulting aptamer-polymer-active agent
bioconjugate device will be adapted to deliver therapeutically effective
amounts of an active agent to targeted leukemia or other cancer cells.

[0074] By using an immobilized TEMPO as the catalyst to oxidize the starch
and form carboxyl groups, activation of the carboxyl group by NHS and EDC
will allow for the binding of an amine-modified aptamer to the surface of
the polymer colloid, thereby forming a covalent linkage. The number of
functional groups on the surface of the nanoparticle may determine the
aptamer surface density.

[0075] TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) reacts with
the hydroxyl groups on the starch polymers in an aqueous medium to create
the desired carboxyl groups (--COOH) by the process known as
TEMPO-mediated carboxylation. NaBr is used to stabilize this reaction.
Hypochlorite (NaClO) initiates the reaction by keeping the pH at
10.2-10.5. Then HCl can be used to lower the pH and reprotonate the
carboxyl groups. 1-Ethyl-3-(3-dimethylaminopropyl) carbodimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are chemicals which
can act as coupling agents to form carboxyl-amino covalent linkages,
which link the carboxylated starch nanoparticle to the 3'-amine-modified
ssDNA aptamer.

[0076] The following examples serve to illustrate one or more parts of one
or more inventions and are not intended to limit any claim.

Example 1

Incorporation of Fluorescent Agents into Starch Based Nanoparticles

[0077] Incorporation of two compounds, in particular the fluorescent model
compound Calcein and the fluorescent anticancer agent doxorubicin (IUPAC
Name: (7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,-
9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,-
12-dione; commercial products include Adriamycin® and Doxil®), into
biopolymer nanoparticles (EcoSphere® 2202 from EcoSynthetix Inc.) was
accomplished by a phase separation technique. This technique involves the
formation of a water-in-oil emulsion. In a 250 mL round bottom flask, the
starch based nanoparticles were dispersed at <5% solids (w/w) in water
under mechanical agitation at a pH of about 10 using dilute caustic. The
resultant dispersion was titrated to a pH of 7 using dilute hydrochloric
acid. The substance to be incorporated in the nanosphere colloid matrix
(calcein or doxorubicin) was dissolved in the dispersion containing the
biopolymer nanoparticles. The amount of encapsulated active agent
prepared ranged from 0.04%-0.4% (w/w). The flask was placed inside an
insulated container and secured properly. The solution was then stirred
for several minutes. Hexane was added dropwise under continuous agitation
until an emulsion was formed. The emulsion was immediately frozen using
liquid nitrogen. The flask was connected to a vacuum system and
lyophilization was carried out at -85° C. After 24 hours, when the
vacuum gauge indicated no further vapor removal, the dried sample was
removed from the vacuum system and stored at -10° C.

Example 2

Drug Release Studies

[0078] The use of fluorescent dyes as spectral probes to investigate
inclusion complexation is known (see Saenger, W. Angew. Chem. 1980, 92,
343-61 and Wenz, G. Angew. Chem. 1994, 106, 851-70). This approach was
adopted in studying the efficacy of starch based nanoparticles (in this
example we used EcoSphere® 2202 from EcoSynthetix Inc.) to
encapsulate selected drugs and the ability of this material to release
the drug over time. Fluorescent compounds such as calcein and doxorubicin
are very sensitive to environmental changes. The fluorescent signal of
the molecules was enhanced when it was incorporated into the matrix of
starch based nanoparticles. As shown in FIG. 8, the signal intensity of
free doxorubicin is much lower than that of the encapsulated doxorubicin.
In addition, a significant hypsochromic shift (change of spectral band
position in the emission spectrum of a molecule to a shorter wavelength)
is observed when doxorubicin is encapsulated. FIG. 9A shows a series of
fluorescence spectra of doxorubicin obtained as a function of time. It
can be seen that there is a significant decrease in signal intensity with
time, indicating sustained release of the active agent. In addition,
there was a relatively small bathochromic shift (change of spectral band
position in the emission spectrum of a molecule to a shorter wavelength)
observed. Without intending to be limited to any theory of operation, it
appears that the reduced shift indicates a biphasic release mechanism
given that not all of the active agent was released over the course of
the 12 hour experiment. FIG. 10A shows a series of fluorescence spectra
of calcein obtained as a function of time. It can be seen that there is a
decrease in signal intensity with time, indicating sustained release of
the active agent.

[0079] The data shown in FIGS. 9A and 10A illustrate that enhancement in
signal intensity for calcein and doxorubicin due to inclusion
complexation with the starch based nanoparticles can be used to monitor
the release of the active agents. FIGS. 9B and 10B are plots of signal
intensity as a function of time at the maximum signal intensity of the
fluorescence emission spectra for doxorubicin and calcein, respectively.
These data show that the concentration of fluorophore molecules inside
the supramolecular cavity is changing with time. The release of the
molecules appears to be proportional to the concentration gradient of the
active agents. The sustained release of active agents from the biopolymer
nanoparticles extended to more than 10 hours. The results demonstrate
that the biopolymer nanoparticles provide a stable matrix for the steady
release of active agents over an extended time period. The release
mechanism appears to be predominantly diffusion controlled.

Example 3

In Vivo Studies of Human Xenographs Implanted in Athymic Mice

[0080] In order to demonstrate the efficacy of the starch based
nanoparticles as a drug delivery device, they were loaded with the
anticancer drug doxorubicin as described in Example 1. The doxorubicin
loaded nanoparticles were administered to athymic mice which had a human
xenograph of a primary brain tumor (D 245 glioblastoma multiform)
previously grown at a subcutaneous site. Athymic mice were chosen for
these studies because normal mice are capable of immunologically
rejecting implanted foreign xenographs, specifically human tumors. The
animals (both control and treated) were monitored for tumor regression
and survival. The results of the study are presented in Table 1.

[0081] The procedure consisted of inoculation of a tumor brei into a
subcutaneous site in athymic mice. The subcutaneous tumors were grown to
approximately 200 cubic millimeters in size (6-8 mm in diameter).
Subsequently, either the free drug or the drug loaded nanoparticles were
injected at the tumor site or i.p. (intra peritoneal). Typically it took
approximately 20 days for the animals to test out. The animals were
treated in groups of 8 to 10 individuals. The highest survival rates
(highest T-C values or increased life span in days) occurred in
individuals in which several doses of doxorubicin loaded nanoparticles
were administered. Table 1 demonstrates the efficacy as well as the
safety of the doxorubicin loaded biopolymer nanoparticles in treating a
primary human brain tumor in athymic mice.

[0082] The nanoparticle may be conjugated to a ligand specific for a
tumor, metastatic cancer cell, or other targeting tissue or organ. This
capability was demonstrated by the following procedures and tests.

[0083] Oxidation of EcoSphere

[0084] Various different types of functionalities may be introduced onto
the starch based nanoparticles to provide binding sites for the aptamer
as well as the active agent. As described in the Detailed Description,
various chemical modification techniques can be employed. A particularly
useful chemical modification is oxidation of starch to produce carboxyl
functionalities. To illustrate this, TEMPO-mediated oxidation was carried
out for both the starch based nanoparticles (EcoSphere® 2202 from
EcoSynthetix Inc.) as well as for regular native (unmodified) corn
starch. In this method, starch was oxidized with sodium hypochlorite
(NaClO) and 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) radicals, at
temperatures between 0 and 4° C. and pH of 10.8. The degree of
oxidation was controlled by amount of NaClO added. As noted, two types of
starch were used. The first was EcoSphere® starch based nanoparticles
and the second one was regular corn starch purchased from Sigma-Aldrich.
The procedures were as described below.

[0085] In a glass jar, 4 g of EcoSphere and 80 mL MilliQ water were added
and mixed thoroughly to create a ˜5% dispersion. In a second jar 4
g of Corn Starch and 80 mL of MilliQ water were added to create a
˜5% solution. The second jar was heated up to above 80° C.
(max 95° C.) under agitation and allowed to fully dissolve.
Subsequently it was cooled to room temperature. Separately, in two 45 mL
tubes 40 mL of water, 38 mg TEMPO, and 508 mg NaBr were added into each
tube (0.01 mol TEMPO per anhydroglucose unit of starch; 0.2 mol NaBr per
anhydroglucose unit of starch), stirred until fully dissolved, and cooled
for 30 minutes in an ice batch. Next the content of one tube was mixed
into each jar. A pH measurement was taken, which initially was 3.8 for
the EcoSphere jar and 7.4 for the Corn Starch jar. Next 450 μL of 0.5
M NaOH was added to the EcoSphere jar to reach pH 10.75, and 200 μL of
0.5 M NaOH was added to the Corn Starch jar to reach pH 10.75.
Subsequently, 10 mL of NaClO was added when the pH dropped to around 6-7,
and pH measurements were taken every 10-15 min. As the mixtures continued
to stir and the pH dropped, the color became darker (yellow/orange). A
total of 60 mL NaClO was added and the pH was finally adjusted to 8.0
before the oxidized starch was diluted 1:1 with ethanol. Ethanol
precipitated the modified EcoSphere nanoparticles and modified starch and
they were harvested by centrifugation and washed by water and ethanol and
finally dried by lyophilization (freeze-drying).

[0086] The oxidized EcoSphere was characterized by zeta-potential measure
and dynamic light scattering. Zeta measurement showed that the modified
particles carried a negative charge with zeta-potential of -25.5 mV,
while unmodified particles were essentially neutral. The size of the
particles appeared to be slightly smaller compared to the non-oxidized
ones (i.e. the NTA Mode was 113 versus 141 nm).

[0087] The color of the final product depended on the pH of the solution
after oxidization. If the pH was too high (higher than 10), a yellow
colored product was obtained. It was found that this color can be removed
by lowering the pH.

[0088] DNA Attachment

[0089] Subsequently, amino-modified and fluorescently labeled DNA was
attached to the starch nanoparticles using
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) as a
coupling agent. The reaction mixture contained 5 μM FAM
(6-carboxyfluorescein) and amino dual labeled DNA, 1-5% COOH-modified
starch, 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6.0
and 20 mM freshly prepared EDC was added the last. Agarose gel
electrophoresis was carried out for DNA and DNA-conjugated to
TEMPO-oxidized EcoSphere nanoparticles. It was found that the gel
fluorescence intensity was more evenly distributed and some of the DNA
migrated more slowly, indicating conjugation to the starch nanoparticles.
In some of the alternative DNA attachment protocols the carboxyl groups
on starch were first activated using N-Hydroxylsuccinimide (NHS) at 5 mM
(1/4 of the amount of EDC) for 15 minutes before adding the DNA. Next
this mixture was allowed to react for several hours. Without intending to
be bound by any particular theory of operation, NHS may help to
facilitate the EDC linking reaction by activating carboxyl groups so it
can react with an amine to form an amide, rather than a salt with an
amine.

[0090] Thus the DNA used in this example, which served as a model compound
for ligand attachment, was successfully attached. The DNA sequence was
5'-FAM-ACG CAT CTG TGA AGA GAA CCT GGG-NH2-3'.

[0091] Attachment of an Aptamer

[0092] An aptamer was attached to EcoSphere® 2202 particles using the
procedure described above. Attachment of the aptamer was confirmed by
laboratory observations of nanoparticle fluorescence. The aptamer was,
AS1411, which is believed to have (as modified) the sequence:
5-Cy3-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-NH2-3' (i.e. AS1411 aptamer with
Cy3 fluorescent tag and amine group). The fluorescent tag, used for
imaging purposes in the diagnostic gel electrophoresis test, can of
course be omitted if needed. However, an additional purpose for the
fluorescent tagging is to facilitate monitoring of the binding and uptake
of the modified nanoparticles by a cell. As for the DNA described above,
the aptamers had an amine modification on the 3' end of the DNA so that
it could be linked using EDC chemistry to carboxyl functionalities on the
nanoparticle.

[0093] Four 200 microliter wells were prepared with cells of an ovarian
cancer cell line (HeLa) and given time to culture and grow. Well 1 was
left with only the HeLa cells. Well 2 had unconjugated AS1411 added to
it. Well 3 had EcoSphere® 2202 nanoparticles with conjugated AS1411
added to it. Well 4 had nanoparticles conjugated with a control sequence
added to it. The control sequence has no known affinity for HeLa cells.
The wells were then allowed to culture for a further 48 hours.

[0094] After the 48 hours had elapsed, cells from the wells were washed to
remove any fluorescent marks on any unbound particles external top the
cells. The cells were then observed under a fluorescence microscope.
Significant fluorescent marks were observed within the cells of well 3
confirming that the nanoparticle/aptamer conjugate had been taken into
the cells.

[0095] Drug Adsorption and Release Studies

[0096] In a dilute aqueous dispersion (e.g. 1-5%) the EcoSphere
nanoparticles are highly swollen and their density is close to that of
water. As a result, centrifugation and even ultracentrifugation were
ineffective methods to separate the particles from the aqueous dispersion
media. Instead, drug loading was evaluated by way of fluorescence change.
It was found that the adsorption of the anticancer drug doxorubicin (Dox)
was very much improved after modification of the EcoSphere nanoparticles
with carboxylate groups. Upon adsorption, the fluorescence of Dox was
also quenched by the carboxylated EcoSphere. This was clearly visible
under the 245 nm excitation in a dark room using a handheld UV lamp. The
fluorescence quenching provides an analytical method to monitor Dox
adsorption.

[0097] To ensure that the observed quenching was not due to a pH effect,
the fluorescence was subsequently compared for the following: Dox was
dissolved at a final concentration of 0.01 mg/mL in unmodified EcoSphere,
COOH-modified EcoSphere and buffer (no EcoSphere). For each condition,
two pH conditions were tested to contain either 20 mM sodium acetate
buffer (pH 5.0) or 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) buffer, pH 7.6. The final pH was confirmed to be at the
intended values.

[0098] Free Dox fluorescence was strong in both pH 5 and 7.6 in water.
Mixing with 1% unmodified EcoSphere nanoparticle dispersion induced about
50% fluorescence quenching but mixing with a COOH-modified EcoSphere
nanoparticle dispersion completely quenched the fluorescence. This
confirmed that COOH-modified EcoSphere is better at adsorbing Dox.
Without intending to be bound by any particular theory of operation, this
is likely due to electrostatic interactions with the positively charged
Dox. Therefore, tuning the EcoSphere charge will allow selective
adsorption of various drugs and in addition provide a way of controlling
the release profile.

[0099] Electrokinetic Measurements

[0100] To evaluate the presence of electrostatic charges on the surface of
the particles, the zeta potential of the biopolymer nanoparticles and
TEMPO oxidized biopolymer nanoparticles was determined from the analysis
of the electrokinetic measurements using a Brookhaven ZetaPlus
instrument. The cross-linked starch particles were suspended in a
solution of NaCl ranging from 0.001 M to 0.1 M concentration, and their
electrophoretic mobilites were determined. The electrophoretic mobilities
were converted to zeta (ξ) potentials using the Smoluchowski
expression, which assumes small particles and dilute ion concentration.
The zeta potential of the un-modified starch based nanoparticles was
determined to be close to zero, whereas the zeta potential of the TEMPO
modified biopolymer nanoparticles was determined to be -25 mV which
indicates negatively charged nanoparticles.

[0101] Particle Size Analysis

[0102] The particle size of dispersed starch based nanoparticles and the
TEMPO modified nanoparticles was determined by Nanoparticle Tracking
Analysis (NTA) using an LM 20 tracking analysis device (NanoSight Ltd.)
equipped with a blue laser (405 nm). The device uses a 50 mW laser
operating in the CW mode to illuminate the particles. The light scattered
by the particles is captured using a digital camera and the motion of
each particle is tracked from frame to frame using NanoSight software. A
high speed video is obtained (30 frames per second, average video about
30 s). The trajectories of individual particles are generated from the
video sequence and the mean squared displacement determined for each
particle. Typically at least 20 trajectories are acquired and 250 to 500
sets of trajectories (each set corresponding to an individual particle)
are accumulated in a video sequence. The analysis of the mean squared
displacement is used to calculate the diffusion coefficient and the
hydrodynamic radius (rh) is determined using the Stokes-Einstein
equation. Thus, the diameter of each particle in the sample can be
determined and a true particle size distribution derived. Because a
diffusion coefficient is obtained for each particle in the field of view,
a particle size distribution can be obtained which does not assume a
particular mathematical model as in dynamic laser light scattering (DLS)
analysis.

[0103] Dispersions of biopolymer nanoparticles were prepared using the
following procedure: 1) dry agglomerate EcoSphere® powder was mixed
in water containing 0.4 wt % sodium carbonate ("lite soda ash") based on
dry weight in a Silverson high shear mixer for 15 minutes; the final
concentration of the dispersed biolatex ranged from 0.015 to 0.030%
(w/w); 2) this dispersion was heated to 45° C. for 15 minutes in a
water bath prior to measurement to ensure the agglomerate particles were
fully dispersed into nanoparticles.

[0104] The above description and attached figures are intended to
illustrate at least one embodiment of each claim and not to limit any
invention. The invention is defined by the following claims.

Patent applications by Aareet Krsna Ganesh Shermon, Waterloo CA

Patent applications by Abdel Rahman Elsayed, Waterloo CA

Patent applications by Ian J. Mclennan, Burlington CA

Patent applications by Juewen Liu, Kitchener CA

Patent applications by Nathan Jones, Hamilton CA

Patent applications by Ryan Wagner, Kitchener CA

Patent applications by Steven Bloembergen, Okemos, MI US

Patent applications by ECOSYNTHETIX LTD.

Patent applications in class PREPARATIONS CHARACTERIZED BY SPECIAL PHYSICAL FORM

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